Transcription factors (TF) are key regulators of gene expression. Most TFs reside in the cytosol and when activated translocate to the nucleus to stimulate gene activity. A therapeutically important TF is NFkB which stimulates genes involved in cell cycling, proliferation and inflammatory responses and is an important molecular target for the development of anti-cancer and anti-inflammatory drugs. Since nuclear translocation is a critical event in NFkB's ability to regulate gene expression, technologies that measure NFkB translocation could be used to screen for novel anti-neoplastic and anti-inflammatory agents that block the translocation process. However, there are few technologies to measure the movement of TFs from the cytosol to the nucleus and none that is amenable for high throughput drug discovery. DiscoveRx has developed an enzyme fragment complementation (EFC) technology that could be used to detect the translocation of TFs using an extremely sensitive beta-galactosidase (B-gal) complementation assay. This method utilizes two genetically-engineered fragments of E. coli Beta-gal. The larger fragment, termed Enzyme Acceptor (EA), contains a deletion near the amino terminus, while the smaller fragment, termed Enzyme Donor (ED), contains the amino-terminal sequence missing from EA. Alone, EA is inactive. However, it can recombine with ED to form an active enzyme that can catalyze the formation of a fluorescent product detected photometrically as a visually amplified response. We propose to develop a direct assay for NFkB translocation using EFC technology. We will engineer ED into NFkB and target EA to the nucleus. When ED-NFkB translocates to the nucleus it will be able to recombine with EA to generate an active Beta-gal that can catalyze the formation of fluorescent products. As a complementary approach to measure NFkB translocation, we will develop a novel NFkB reporter gene assay using our EFC technology in which an NFkB response element drives the production of a GST-ED fusion protein whose expression will be detected with EA in a simplified cell based fluorescent assay. These studies will develop the first NFkB translocation for drug discovery. In the future, we will adapt this assay as a general technology for drug discovery against any TF to open up an entirely new field of transcription factor Pharmacology and therapeutics.